Limits...
Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

No MeSH data available.


Related in: MedlinePlus

(a) Swarms of RP6894 (ΔmotAB) cells harboring plasmids that express MotA/B(A39V) (A39V), MotA(M206G)/B(A39V) (A39V/M206G), MotA(M206A)/B(A39V) (A39V/M206A), MotA(M206S)/B(A39V) (A39V/M206S) or MotA(M206T)/B(A39V) (A39V/M206T). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 20 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express PomA/B (WT), PomA/B(C31A) (C31A), PomA/B(L28A/C31A) (L28A/C31A) or PomA(L183F)/B(L28A/C31A) (L28A/C31A/L183F). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG10 (1% polypeptone, 0.4% K2HPO4, 10 mM NaCl, 490 mM KCl, 0.5% glycerol) and 0.25% agar. Plates were incubated in the presence of 0.04% arabinose and 2.5 μg/ml chloramphenicol at 30°C for 12 hr. Vec. means a pBAD33 plasmid vector. (d) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036635&req=5

f5-5_45: (a) Swarms of RP6894 (ΔmotAB) cells harboring plasmids that express MotA/B(A39V) (A39V), MotA(M206G)/B(A39V) (A39V/M206G), MotA(M206A)/B(A39V) (A39V/M206A), MotA(M206S)/B(A39V) (A39V/M206S) or MotA(M206T)/B(A39V) (A39V/M206T). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 20 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express PomA/B (WT), PomA/B(C31A) (C31A), PomA/B(L28A/C31A) (L28A/C31A) or PomA(L183F)/B(L28A/C31A) (L28A/C31A/L183F). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG10 (1% polypeptone, 0.4% K2HPO4, 10 mM NaCl, 490 mM KCl, 0.5% glycerol) and 0.25% agar. Plates were incubated in the presence of 0.04% arabinose and 2.5 μg/ml chloramphenicol at 30°C for 12 hr. Vec. means a pBAD33 plasmid vector. (d) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.

Mentions: Blair and coworkers deduced the arrangement of core membrane segments of the MotA/B complex in E. coli using systematic cysteine-scanning mutagenesis and tryptophan replacement techniques7,8,20,21. On the basis of those reports, the side chain of Met206 in MotA (which corresponds to Leu183 in PomA) is thought to be located opposite to Ala39 in the channel. Here, randomized mutations were introduced in M206 of MotA in the A39V MotB mutant background. We chose A39V because valine is the smallest residue with non-motile behavior (Figures 2 and 3). As shown in Figure 5a, the loss of function caused by the A39V mutation was rescued by an additional mutation in MotA (M206S, M206G, M206A or M206T), and the increase of volume at position 39 by Val (50 Å3, compared to Ala) is close to the decrease of volume at position 206 by the substitution to Ser (72 Å3, compared to Met). Although volume difference between Met and Thr was much closer to the difference between Met and Ser, the recovery of the function by Met to Thr replacement is not so much. Therefore the orientation of the side chain of introduced amino acids may also be important for the ion flux. The protein expression level of MotA was not affected by any mutation at M206, although that of MotB was slightly affected by the mutations (Figure 5b). Similar experiments were carried out for PomA/B, and similar results were obtained. As shown in Figure 5c, the PomB(L28A/C31A) double mutant shows a slow-motile phenotype, whereas the function was rescued by an additional mutation of PomA(L183F). We confirmed that the protein expression level of PomA and PomB was not affected by any of the mutations (Figure 5d). Thus, we demonstrate here that Cys31 of PomB and Ala39 of MotB are located at the ion flux pathways, facing Leu183 of PomA (for PomB) and Met206 of MotA (for MotB) in the channel pores of the PomA/B and MotA/B stator units of the flagellar motors.


Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors
(a) Swarms of RP6894 (ΔmotAB) cells harboring plasmids that express MotA/B(A39V) (A39V), MotA(M206G)/B(A39V) (A39V/M206G), MotA(M206A)/B(A39V) (A39V/M206A), MotA(M206S)/B(A39V) (A39V/M206S) or MotA(M206T)/B(A39V) (A39V/M206T). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 20 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express PomA/B (WT), PomA/B(C31A) (C31A), PomA/B(L28A/C31A) (L28A/C31A) or PomA(L183F)/B(L28A/C31A) (L28A/C31A/L183F). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG10 (1% polypeptone, 0.4% K2HPO4, 10 mM NaCl, 490 mM KCl, 0.5% glycerol) and 0.25% agar. Plates were incubated in the presence of 0.04% arabinose and 2.5 μg/ml chloramphenicol at 30°C for 12 hr. Vec. means a pBAD33 plasmid vector. (d) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036635&req=5

f5-5_45: (a) Swarms of RP6894 (ΔmotAB) cells harboring plasmids that express MotA/B(A39V) (A39V), MotA(M206G)/B(A39V) (A39V/M206G), MotA(M206A)/B(A39V) (A39V/M206A), MotA(M206S)/B(A39V) (A39V/M206S) or MotA(M206T)/B(A39V) (A39V/M206T). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing TG and 0.26% agar. Plates were incubated in the presence of 0.04% arabinose at 30°C for 20 hr. Vec. means a pBAD24 plasmid vector. (b) Western blotting analysis of MotA (left panel) and MotB (right panel) in whole cell extracts. Proteins were detected with an anti-MotA266 antibody and an anti-MotB2 antibody. (c) Swarms of NMB191 (ΔpomAB) cells harboring a plasmid that express PomA/B (WT), PomA/B(C31A) (C31A), PomA/B(L28A/C31A) (L28A/C31A) or PomA(L183F)/B(L28A/C31A) (L28A/C31A/L183F). Transformants were cultured in liquid medium overnight, and aliquots were spotted onto a plate containing VPG10 (1% polypeptone, 0.4% K2HPO4, 10 mM NaCl, 490 mM KCl, 0.5% glycerol) and 0.25% agar. Plates were incubated in the presence of 0.04% arabinose and 2.5 μg/ml chloramphenicol at 30°C for 12 hr. Vec. means a pBAD33 plasmid vector. (d) Western blotting analysis of PomA (left panel) and PomB (right panel) in whole cell extracts. Proteins were detected with an anti-PomA1312 antibody and an anti-PomB93 antibody.
Mentions: Blair and coworkers deduced the arrangement of core membrane segments of the MotA/B complex in E. coli using systematic cysteine-scanning mutagenesis and tryptophan replacement techniques7,8,20,21. On the basis of those reports, the side chain of Met206 in MotA (which corresponds to Leu183 in PomA) is thought to be located opposite to Ala39 in the channel. Here, randomized mutations were introduced in M206 of MotA in the A39V MotB mutant background. We chose A39V because valine is the smallest residue with non-motile behavior (Figures 2 and 3). As shown in Figure 5a, the loss of function caused by the A39V mutation was rescued by an additional mutation in MotA (M206S, M206G, M206A or M206T), and the increase of volume at position 39 by Val (50 Å3, compared to Ala) is close to the decrease of volume at position 206 by the substitution to Ser (72 Å3, compared to Met). Although volume difference between Met and Thr was much closer to the difference between Met and Ser, the recovery of the function by Met to Thr replacement is not so much. Therefore the orientation of the side chain of introduced amino acids may also be important for the ion flux. The protein expression level of MotA was not affected by any mutation at M206, although that of MotB was slightly affected by the mutations (Figure 5b). Similar experiments were carried out for PomA/B, and similar results were obtained. As shown in Figure 5c, the PomB(L28A/C31A) double mutant shows a slow-motile phenotype, whereas the function was rescued by an additional mutation of PomA(L183F). We confirmed that the protein expression level of PomA and PomB was not affected by any of the mutations (Figure 5d). Thus, we demonstrate here that Cys31 of PomB and Ala39 of MotB are located at the ion flux pathways, facing Leu183 of PomA (for PomB) and Met206 of MotA (for MotB) in the channel pores of the PomA/B and MotA/B stator units of the flagellar motors.

View Article: PubMed Central - PubMed

ABSTRACT

Flagellar motor proteins, MotA/B and PomA/B, are essential for the motility of Escherichia coli and Vibrio alginolyticus, respectively. Those complexes work as a H+ and a Na+ channel, respectively and play important roles in torque generation as the stators of the flagellar motors. Although Asp32 of MotB and Asp24 of PomB are believed to function as ion binding site(s), the ion flux pathway from the periplasm to the cytoplasm is still unclear. Conserved residues, Ala39 of MotB and Cys31 of PomB, are located on the same sides as Asp32 of MotB and Asp24 of PomB, respectively, in a helical wheel diagram. In this study, a series of mutations were introduced into the Ala39 residue of MotB and the Cys31 residue of PomB. The motility of mutant cells were markedly decreased as the volume of the side chain increased. The loss of function due to the MotB(A39V) and PomB(L28A/C31A) mutations was suppressed by mutations of MotA(M206S) and PomA(L183F), respectively, and the increase in the volume caused by the MotB(A39V) mutation was close to the decrease in the volume caused by the MotA(M206S) mutation. These results demonstrate that Ala39 of MotB and Cys31 of PomB form part of the ion flux pathway and pore with Met206 of MotA and Leu183 of PomA in the MotA/B and PomA/B stator units, respectively.

No MeSH data available.


Related in: MedlinePlus